The present invention relates to a process for making lipid membrane structures (i.e., vesicles or liposomes; referred to herein as "vesicles") in an effective, efficient and reproducible manner.
Vesicles are microscopic globules, having a maximum diameter on the order of about 10,000 A and preferably having a diameter between about 300 and about 2,000 A, bounded by a wall formed by one or more bimolecular layers (bilayers) of a compound containing a hydrophilic polar group and a hydrophobic non-polar group. The vesicles contain an aqueous liquid, for example an aqueous solution of a biologically-active substance, and generally exist in a colloidal dispersion in an aqueous medium, such as a saline solution. Vesicles provide a method for encapsulating aqueous liquids and are particularly useful for administering biologically-active substances to living organisms, while avoiding the destruction or deactivation of those substances, for example, in the bloodstream, before the substances reach their site of biological activity in the body. Thus, EDTA has been encapsulated in vesicles and injected as treatment for heavy metal poisoning; Rahman, et al., J. Lab. Clin. Med., 83 (4), 640-647 (1974), and U.S. Pat. No. 3,932,657, issued Jan. 13, 1976 and assigned to the U.S. Energy Research and Development Administration. Liposomes containing insulin have been disclosed for oral delivery; Patel, et al., FEBS Letters, 62, 1, 60-63 (1976) and South African Pat. No. 73/1850. Actinomycin D has been encapsulated in liposomes and used in cancer chemotherapy; Proceedings of The Society for Experimental Biology and Medicine, 146, 1173-1176 (1974). Vesicles targeted to the liver through the use of digalactosyl diglyceride moieties, containing pharmaceutical agents such as insulin or interferon, have also been disclosed; U.S. patent application Ser. No. 75,310, Geho, filed Sept. 13, 1979, incorporated herein by reference. The New England Journal of Medicine, Sept. 23, 1976, pages 704-710 and Sept. 30, 1976, pages 765-770, contains an extensive report on liposomes, their use in delivering drugs, and includes various references to the types of pharmaceutical agents which have been encapsulated in liposomes.
The art discloses at least three types of processes for making vesicles: injection, sonication, and dialysis. Each one has significant disadvantages in terms of making well-defined vesicles having controlled physical/chemical properties, and/or in scaling up to produce commercial quantities of vesicles. In the sonication process, the lipid material is dissolved in an organic phase and the organic phase is then removed, producing a thin lipid film, the aqueous phase is added to this, and, finally, ultrasonic energy is added to the system. See Huang, Biochemistry, 8, 344 (1969); and U.S. patent application Ser. No. 75,310, Geho, filed Sept. 13, 1979, all of which are incorporated herein by reference. Such processes are difficult to reproduce, require the application of high energy to the vesicle system, and yield vesicles having wide variations in their physical properties (e.g., size and trapped volume). Obviously, when vesicles are prepared as a dosage form pharmaceutical composition, such factors can be of critical importance. In the dialysis process, lipid materials are dissolved in a detergent, e.g., sodium cholate, and vesicles are formed as the detergent is removed by dialysis. Only a restricted class of detergents, i.e., bile salts, are useful in the dialysis process and these detergents are very difficult to remove completely from the final product. Further, the process is slow and poorly suited to commercial scale-up. See Rhoden and Goldin, Biochemistry, 18, 4173 (1979), incorporated herein by reference. In the injection process, the lipid material in an organic phase is injected through a syringe into an aqueous phase. See Batzri, et al., Biochemica et Biophysica Acta, 298, 1015-1019 (1973); Deamer, et al., Biochemica et Biophysica Acta, 443, 629-634 (1976); and Kremer, et al., Biochemistry, 16, 3932-3935 (1977), all of which are incorporated herein by reference. Such processes are very difficult to scale up commercially and, further, require relatively harsh reaction conditions (i.e., high agitation and temperature) which can be detrimental to the pharmaceutically-active material being encapsulated.
The process of the present invention is a modified injection process and permits the manufacture of vesicles while providing a vast array of benefits over these prior art processes. Specifically, the process of the present invention provides:
(a) a method for making vesicles under gentle conditions (i.e., below the transition temperature of the lipid materials, and without requiring high agitation). PA1 (b) vesicles which trap the aqueous phase efficiently and maintain their contents effectively; PA1 (c) vesicle dispersions exhibiting good colloidal stability; PA1 (d) vesicles having easily-reproduced physical properties; and PA1 (e) a method for producing vesicles which is continuous and easily scalable to commercial levels. PA1 (a) S.sub.1 is highly soluble in the aqueous solution; PA1 (b) S.sub.2 is hydrophobic; PA1 (c) S.sub.2 is more volatile than the aqueous solution; PA1 (d) the membrane components are not entirely soluble in S.sub.2 alone; PA1 (e) the mixture of S.sub.1 and S.sub.2 forms an interface with the aqueous solution; and PA1 (f) the membrane components can be dissolved in a mixture of S.sub.1 and S.sub.2. PA1 (a) S.sub.1 and S.sub.2 must be selected such that together they dissolve the membrane materials, for example, in the form of either single molecules, micelles, inverted micelles, or as a microemulsion at room temperature and pressure (or, more particularly, at the temperature/pressure at which the process is to be carried out). Where the membrane material used in the process is distearoyl lecithin or dipalmitoyl lecithin, S.sub.1 cannot be methyl acetate or ether, based on this criterion. PA1 (b) The organic phase (the mixture of S.sub.1 and S.sub.2), in the absence of the membrane materials, must not, in the proportions used in the process, be entirely soluble in the aqueous phase, i.e., the organic phase must form an interface with the aqueous phase. PA1 (c) S.sub.1 must be highly soluble in the aqueous phase. Solubility can be measured by the partition coefficient, which is equal to the ratio of the amount of S.sub.1 going into the aqueous phase: the amount of S.sub.1 going into S.sub.2 assuming both phases have equal volumes, at room temperature and pressure (or, more particularly, at the temperature pressure at which the process is to be carried out). To be useful in the present invention, the partition coefficient for S.sub.1 must be greater than about 0.1 (i.e., more than 10% of S.sub.1 must be soluble in the aqueous phase), preferably it is greater than about 0.5 and most preferably greater than about 10. Based on this criterion, S.sub.1 cannot be pentanol or higher alkanols under normal temperature and pressure conditions. PA1 (d) S.sub.2 must be hydrophobic, i.e., it must form an interface with the aqueous solution. PA1 (e) S.sub.2 must be more volatile than the aqueous phase, in order to permit it to be stripped selectively from the aqueous phase at a latter stage in the process. PA1 (f) S.sub.2, by itself, must not entirely dissolve the membrane material. As a result of this criterion, chloroform cannot be used for S.sub.2 where the membrane material is distearoyl or dipalmitoyl lecithin. PA1 (1) radionuclides, especially technecium-99m, thallium-201, indium-113m, indium-111, fluorine-18, strontium-85 and iodine-125; PA1 (2) heavy metal chelators, especially the ethylene diamine tetraacetates and the diethylene triamine pentaacetates; PA1 (3) insulin, or insulin derivatives; PA1 (4) antiviral agents, such as those used in the treatment of hepatitis; PA1 (5) interferon; PA1 (6) hormones, e.g., estrogens, glucagon, and catecholamines; PA1 (7) essential amino acids; and PA1 (8) nucleotides (e.g., ATP).
It is, therefore, an object of the present invention to provide an efficient and effective process, having the advantages ennumerated above, for producing vesicles, especially vesicles containing pharmaceutically-active materials.